BU-3862T antitumor antibiotic

ABSTRACT

A new antitumor antibiotic designated herein as BU-3862T is produced by fermentation of Streptomyces hygroscopicus ATCC 53709. BU-3862T and its diacetyl and dihydro derivatives inhibit the growth of tumors in experimental animals.

This is a division of our prior co-pending application Ser. No. 165,337filed 03/07/88, now U.S. Pat. No. 4,912,133.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to novel antitumor antibiotics and to theirproduction and recovery.

2. Description of the Prior Art

The present invention provides a novel fermentation product designatedBU-3862T (I) which has the structure ##STR1## Also provided are thediacetate (II) and dihydro (III) derivatives of BU-3862T which have thestructures shown below. ##STR2##

Applicants are not aware of any antitumor antibiotics related instructure to the compounds of the present invention.

SUMMARY OF THE INVENTION

This invention relates to a new antitumor antibiotic designated hereinas BU-3862T which has the structural formula ##STR3## and to the processfor the preparation, isolation and purification of BU-3862T.

The antibiotic of the present invention is obtained by fermentation of aBU-3862T-producing strain of Streptomyces hygroscopicus, preferablyStreptomyces hygroscopicus strain P247-71 (ATCC 53709) or a mutant orvariant thereof, in an aqueous nutrient medium under submerged aerobicconditions until a substantial amount of BU-3862T is produced by saidorganism in said culture medium and, optionally, recovering the BU-3862Tfrom the culture medium.

Also provided are the diacetyl derivative of BU-3862T having the formula##STR4## wherein Ac═CH₃ CO-- which is produced by acetylation ofBU-3862T and the dihydro derivative of BU-3862T having the formula##STR5## produced by catalytic hydrogenation of BU-3862T.

BU-3862T and its diacetyl and dihydro derivatives exhibit inhibitoryactivity against experimental animal tumor systems, e.g. B16 melanoma inmice.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents the infrared absorption spectrum of BU-3862T (KBr).

FIG. 2 represents the proton magnetic resonance spectrum of BU-3862T inCDCl₃ (400 MHz).

FIG. 3 represents the ¹³ C NMR spectrum of BU-3862T in CDCl₃ (100 MHz).

DETAILED DESCRIPTION

The BU-3862T antibiotic of the present invention is produced byfermentation of a BU-3862T-producing strain of Streptomyceshygroscopicus.

A preferred BU-3862T-producing strain designated strain P247-71 wasisolated from a soil sample collected near the root of a tamarind at Mt.Apo, Davao, Mindanao Island, the Philippines. A biologically pureculture of this strain has been deposited in The American Type CultureCollection, Rockville, Md.; and added to their permanent collection ofmicroorganisms as ATCC 53709.

The results of taxonomic studies performed on strain P247-71 indicatethat the strain belongs to the genus Streptomyces and to the speciesgroup Streptomyces hygroscopicus.

Strain P247-71 has the following properties:

MORPHOLOGY

Both substrate and aerial mycelia are formed. They are long,well-branched and not fragmented into short filaments. Chains ofarthrospores are born on the aerial hyphae. The spore chains and sporemorphology are as follows: 1) spiral spore chains with 2 to 8 turns, 2)monopodially branched sporophores, 3) spores, oval or barrel-shaped (0.5to 0.7 by 0.5 to 1.2 μm), and 4) spore ornamentation, rugose or smooth.

Sporangium, motile spore and sclerotium are not observed.

CULTURAL AND PHYSIOLOGICAL CHARACTERISTICS

Strain P247-71 grows well in most descriptive media. Gray aerialmycelium with hygroscopic black patches is observed on ISP agar mediaexcept for ISP No. 6 medium. White to pale yellowish-gray aerialmycelium is formed on Czapek's sucrose-nitrate agar. The substratemycelium is colorless or yellowish brown to grayish yellow. Melanin andother diffusible pigments are not produced. Most sugars are utilized forgrowth. The cultural and physiological characteristics are shown inTables 1 and 2, respectively.

The morphological, cultural and physiological characteristics of strainP247-71 indicate that the strain belongs to the genus Streptomyces.According to the descriptions of Pridham and Tresner¹, the majorcharacteristics of the strain are summarized as follows: 1) gray aerialmycelium, 2) spiral spore chain, 3) absent melanoid, and 4) smooth sporewall ornamentation. The hygroscopic change of sporulated aerial myceliumis a distinct property of the strain. The major characteristics andthose shown in Tables 1 and 2 of strain P247-71 place it in Streptomyceshygroscopicus.

It is to be understood that the present invention is not limited to useof the particular preferred strain P247-71 described above or toorganisms fully answering the above descriptions. It is especiallyintended to include other BU-3862T-producing variants or mutants of thesaid organism which can be produced by conventional means such as x-rayradiation, ultraviolet radiation, treatment with nitrogen mustards,phage exposure and the like.

                                      TABLE 1                                     __________________________________________________________________________    Cultural characteristics of strain P247-71                                                                    Substrate Diffusible                          Medium       Growth                                                                              Aerial mycelium                                                                            mycelium  pigment                             __________________________________________________________________________    Sucrose-nitrate agar                                                                       Moderate                                                                            Moderate; white to pale                                                                    Strong yellowish                                                                        None                                (Czapek-Dox agar)  yellowish gray                                                                             brown (74)                                    Tryptone-yeast extract                                                                     Moderate;                                                                           None         Colorless None                                broth (ISP No. 1)                                                                          not turbid,                                                                   floccose                                                         Yeast extract-malt                                                                         Good  Scant; light gray (264) to                                                                 Pale orange                                                                             None                                extract agar (ISP No. 2)                                                                         black. Hygroscopic                                                                         yellow (70)                                   Oat meal agar (ISP No. 3)                                                                  Poor  Poor; light gray (264)                                                                     Greenish gray (155)                                                                     None                                Inorganic salts-starch                                                                     Moderate                                                                            Moderate; light gray (264)                                                                 Colorless None                                agar (ISP No. 4)                                                              Glycerol-asparagine agar                                                                   Moderate                                                                            Moderate; brownish                                                                         Colorless None                                (ISP No. 5)        black (65). Hygroscopic                                    Peptone-yeast extract-                                                                     Poor  None         Colorless None                                iron agar (ISP No. 6)                                                         Tyrosine agar (ISP No. 7)                                                                  Moderate                                                                            Moderate; dark gray (266)                                                                  Dark grayish                                                                            None                                                                yellow (91)                                   Glucose-asparagine agar                                                                    Moderate                                                                            Moderate; brownish                                                                         Colorless None                                                   black (65). Hygroscopic                                    Bennett's agar                                                                             Moderate                                                                            Moderate; black.                                                                           Grayish yellow (90)                                                                     None                                                   Hygroscopic                                                __________________________________________________________________________     Observation after incubation at 28° C. for 3 weeks.                    Color and number in parenthesis follow ISCCNBS designation.              

                  TABLE 2                                                         ______________________________________                                        Physiological characteristics of strain P247-71                               ______________________________________                                        Hydrolysis of:             Utilization of:*                                   Gelatin       +            Glycerol    +                                      Starch: Soluble starch                                                                      +            D-Arabinose ±                                   Potato starch +            L-Arabinose +                                                                 D-Xylose    ±                                   Milk coagulation                                                                            -            D-Ribose    +                                      peptonization +            L-Rhamnose  +                                      Production of:             D-Galactose +                                      Nitrate reductase                                                                           -            D-Fructose  +                                      Tyrosinase    -            D-Mannose   -                                                                 L-Sorbose   -                                      Tolerance to:              Sucrose     +                                      Lysozyme, 0.01% (w/v)                                                                       -            Lactose     +                                      0.001% (w/v)  -            Cellobiose  +                                      NaCl, 1%-6% (w/v)                                                                           +            Melibiose   +                                      7% (w/v)      -            Trehalose   ±                                   pH 5.5-10.5   +            Raffinose   +                                      5.0 and 11.0  -            D-Melezitose                                                                              -                                                                 Soluble starch                                                                            +                                      Temperature:               Cellulose   -                                      Growth range  20° C.-39° C.                                                                Dulcitol    -                                      No growth     17° C. and 41° C.                                                            Inositol    +                                      Optimal growth                                                                              37° C.-39° C.                                                                D-Mannitol  +                                                                 D-Sorbitol  -                                                                 Salicin     +                                      ______________________________________                                         *Basal medium: PridhamGottlieb's medium (= ISP No. 9 medium).            

PREPARATION OF BU-3862T

BU-3862T may be produced by cultivating a BU-3862T-producing strain ofStreptomyces hygroscopicus, preferably a strain having thecharacteristics of Streptomyces hygroscopicus strain P247-71 (ATCC53709), or a variant or mutant thereof, under submerged aerobicconditions in an aqueous nutrient medium. The organism is grown in anutrient medium containing an assimilable carbon source, e.g. glycerol,D-ribose, L-rhamnose, D-glucose, D-fructose, sucrose, lactose,melibiose, D-mannitol or soluble starch. The nutrient medium should alsocontain an assimilable nitrogen source such as fish meal, peptone,soybean flour, peanut meal, cottonseed meal or corn steep liquor.Nutrient inorganic salts can also be incorporated in the medium. Suchsalts may comprise any of the usual salts capable of providing sodium,potassium, ammonium, calcium, phosphate, sulfate, chloride, bromide,nitrate, carbonate or like ions.

Production of BU-3862T can be effected at any temperature conducive tosatisfactory growth of the organism, e.g. 20° C. to 39° C., and isconveniently carried out at a temperature of about 28° C.

The fermentation may be carried out in flasks or in laboratory orindustrial fermentors of various capacities. When tank fermentation isto be used, it is desirable to produce a vegetative inoculum in anutrient broth by inoculating a small volume of the culture medium witha slant or soil culture or a lyophilized culture of the organism. Afterobtaining an active inoculum in this manner, it is transferredaseptically to the fermentation tank medium for large scale productionof BU-3862T. The medium in which the vegetative inoculum is produced canbe the same as, or different from, that utilized in the tank as long asit is such that a good growth of the producing organism is obtained.

In general, optimum production of BU-3862T is achieved after incubationperiods of about four days.

BU-3862T may be recovered from the culture medium and isolated in asubstantially pure form by conventional solvent extraction andchromatographic procedures. Example 2 below illustrates a suitableisolation and purification procedure.

The diacetate derivative (II) of BU-3862T may be prepared by reactingBU-3862T with a conventional acetylating agent such as acetic anhydridein an inert organic solvent. A typical procedure is illustrated inExample 3 below. The dihydro derivative (III) of BU-3862T may beprepared by catalytic hydrogenation of BU-3862T as illustrated inExample 4.

BU-3862T was obtained as a colorless sticky solid. It was readilysoluble in dimethyl sulfoxide, dimethylformamide, methanol, ethanol,ethyl acetate and chloroform, but practically insoluble in water,benzene and other organic solvents.

BU-3862T showed positive reaction to iodine, ammonium molybdate-sulfuricacid (AMS) and Rydone-Smith reagents, while it was negative toninhydrin, anthrone and ferric chloride tests. The physico-chemicalproperties of BU-3862T are summarized in Table 3. This compound did notshow characteristic UV absorption. The IR, ¹ H-NMR and ¹³ C-NMR spectraof BU-3862T are illustrated in FIGS. 1, 2 and 3, respectively.

                  TABLE 3                                                         ______________________________________                                        Physico-chemical properties of BU-3862T                                       ______________________________________                                        Nature:   Colorless sticky solid                                              [α].sub.D.sup.24 :                                                                +32 ±2° C. (c 0.5, methanol)                              EI & FDMS:                                                                              m/z 399 (M + H).sup.+                                               Microanalysis:                                                                          Calcd for C.sub.20 H.sub.34 N.sub.2 O.sub.6 : C 60.28, H 8.60,                N 7.03                                                                        Found: C 60.18, H 8.82, N 6.60                                      TLC, SiO.sub.2 :                                                                        CH.sub.2 Cl.sub.2 --MeOH (9:1) Rf 0.37                              (Merck F.sub.254)                                                                       EtOAc-13 MeOH (4:1) 0.59                                                      Methylethylketone-Xylene-MeOH (10:10:2) 0.35                        ______________________________________                                    

STRUCTURAL STUDIES ON BU-3862T

BU-3862T exhibits strong absorptions at 3300 (hydroxy), 1720 (carbonyl),1650 and 1530 cm⁻¹ (amide) in the IR spectrum indicating a peptidestructure for the antibiotic. The ¹³ C-NMR spectrum demonstrated 20carbons which were identified as two C--CH₃, one ═C--CH₃, eight --CH₂,three --CH, one >C<, one >C═CH₂ and three C═O carbons. The molecularformula of BU-3862T was established as C₂₀ H₃₄ N₂ O₆ based on themicroanalysis, mass spectral data ((M+H)⁺ : m/z 399) and ¹³ C-NMRspectral analysis. Thirty-four protons were observed in the ¹ H-NMRspectrum. The two doublet protons (δ7.03 and 6.48 ppm, in CDCl₃)gradually disappeared by the addition of deutrium oxide were assigned totwo --NH--CO groups. The broad two singlet protons (δ4.83 and 4.79) andthe AB type doublet protons (δ3.35 and 3.12, J: 5.0 Hz) were assigned toexomethylene(--(CH₃)C═CH₂) and epoxide ##STR6## protons, respectively.The connectivities of these protons were determined by ¹ H-¹ H COSYexperiments leading to the partial structures as shown below. Furtherconnectivities of the partial structures were established by the ¹³ C-¹H COSY and ¹³ C-¹ H long range COSY experiments. They were analyzed asshown below and thus the total structure of BU-3862T was determined.Further evidence for the structure was provided by its mass spectrum anddegradation experiment. The EI-MS spectrum exhibited abundant fragmentions at m/z 127 (iso-octanoyl), 214 (iso-octanoyl-seryl) and 325(iso-octanoyl-seryl-4,5-didehydroleucyl) supporting the structure. Uponacid hydrolysis, BU-3862T gave an amino acid and a fatty acid. The aminoacid isolated was identified as L-serine by HPLC and the fatty acid asiso-octanoic acid by gas chromatography of its methyl ester. BU-3862Tafforded the diacetate derivative upon treatment with acetic anhydridein pyridine. When hydrogenated over palladium carbon, BU-3862T affordedtwo reduction products, dihydro-BU-3862T and tetrahydro-BU-3862T, whosestructures were determined based on their spectral data. The diacetateand the dihydro compound retained the biological activity but thetetrahydro derivative was devoid of the activity.

BU-3862T may be named1,2-epoxy-2-hydroxymethyl-4-(N-isooctanyl-L-serylamino)-6-methyl-hept-6-ene-3-oneand is a unique peptide containing an epoxide and an exomethylene group.##STR7##

BIOLOGICAL ACTIVITY OF BU-3862T

BU-3862T and diacetyl-, dihydro- and tetrahydro-BU-3862T were tested forin vitro cytotoxicity against several murine and human tumor cell linesand/or for in vivo antitumor activity in mice. Mitomycin C was used as areference compound in both in vitro and in vivo experiments. B16-F10(murine melanoma), P388 (murine leukemia), L1210 (murine leukemia) andMoser (human colorectal carcinoma) cells were grown to the logarithmicphase in enriched Eagle minimum essential medium (MEM) supplemented withfetal calf serum (FCS, 10%) and kanamycin (60 mcg/ml), and HCT-116(human colon carcinoma) cells in Maccoy's 5A medium supplemented withFCS (10%), penicillin (100 u/ml) and streptomycin (100 mcg/ml),harvested and inoculated into wells of the 96- or 24-well tissue cultureplate with test materials at the concentrations of 1.5×10⁵, 1.2×10⁴,1.2×10⁴, 2.5×10⁵ and 3.0×10⁵ cells/ml, respectively. They were incubatedat 37° C. in humidified atmosphere of 5% CO₂ and 95% air for 72 hours.The cytotoxic activities against B16-F10, Moser and HCT-116 cells werecolorimetrically determined at 540 nm after staining viable cells with0.006% neutral red solution. On the other hand, the cytotoxic activitiesagainst P388 and L1210 cells were determined by counting the number ofviable cells. The results were summarized in Table 4. Compared withmitomycin C, BU-3862T showed much more potent cytotoxicity against bothmurine and human cells. The potency was approximately 50-120 timesgreater than that of mitomycin C in terms of IC₅₀ values. The diacetyland dihydro derivatives also showed equivalently potent cytotoxicpotentials against both murine and human cells, approximately half thoseof BU-3862T. On the other hand, the tetrahydro derivative wassignificantly less active than the above compounds.

Inhibitory effects of BU-3862T on macromolecule (DNA, RNA and protein)synthesis were determined in cultured B16-F10 melanoma cells. B16-F10cells (10⁵ cells/ml) were incubated with BU-3862T at 37° C. for 3.5 (forDNA synthesis) or 4 (for RNA and protein synthesis) hours. Isotopicallylabelled precursor, ³ H-thymidine, ¹⁴ C-uridine or ³ H-leucine was addedinto the cultured mixture and further incubated for 30 min (for DNAsynthesis) or 60 min (for RNA and protein synthesis). After washing withchilled 5% trichloroacetic acid solution, the radioactivity incorporatedinto the acid-insoluble fraction of the tumor cells was determined in aliquid scintillation counter. As shown in Table 5, BU-3862T inhibitedboth DNA and protein synthesis similarly and the potency was over 100times higher than that on RNA synthesis in terms of IC50 value.

In vivo antitumor activities of BU-3862T and the diacetyl and dihydroderivatives were determined in tumor-bearing BDF₁ or CDF₁ mice. MaleBDF₁ mice were intraperitoneally inoculated with 0.5 ml of 10% melanoticmelanoma B16 brei and female CDF₁ mice were also intraperitoneallyinoculated with 0.4 ml of diluted ascitic fluid containing 10⁵ lymphoidleukemia L1210 cells or 10⁶ lymphocytic leukemia P388 cells. Testcompounds were intraperitoneally administered to the mice by thefollowing four different treatment schedules; once a day on days 1, 2and 3 (QD×3), on days 1, 4 and 7 (Q3D×3), on days 1, 5 and 9 (Q4D×3) andon days 1 to 9 (QD×9). As shown in Table 6, BU-3862T demonstrated anexcellent therapeutic efficacy against B16 melanoma. When administeredby the Q4D×3 treatment schedule, the potency (minimum effective dose) ofBU-3862T was the same as that of mitomycin C. This compound showedbetter antitumor activity and broader therapeutic range by theintermittent dosing schedule (Q4D×3) than by the consecutive dosingschedule (QD×9) in terms of maximum T/C value and chemotherapeutic index(ratio of optimal dose to minimum effective dose), respectively. Boththe diacetyl and dihydro derivatives also exhibited significant anti-B16melanoma activity by the Q4D×3 treatment schedule but were approximatelyten times less active than the parent compound in terms of minimumeffective dose as shown in Table 7. On the other hand, anti-leukemicactivities of BU-3862T were rather weak. This compound gave moderateantitumor activity against L1210 leukemia with maximum T/C value of 145%and showed no significant prolongation of lifespan in P388leukemia-bearing mice at the doses tested (Tables 8 and 9).

                  TABLE 4                                                         ______________________________________                                        In vitro cytotoxicities aainst murine and human tumor cells                            IC.sub.50 (mc/ml)                                                    Comound    B16-F10  P388    L1210 HCT-116                                                                              Moser                                ______________________________________                                        BU-3862T   0.0017   0.031   0.01  0.0097 0.016                                Diacetyl-  0.0030    ND*    ND    0.017  0.044                                BU-3862T                                                                      Dihydro-   0.0032   ND      ND    0.013  0.038                                BU-3862T                                                                      Tetrahydro-                                                                              0.53     ND      ND    >1.0   >1.0                                 BU-3862T                                                                      Mitomycin C                                                                              0.50     ND      ND    0.80   1.2                                  ______________________________________                                         *ND: Not determined                                                      

                  TABLE 5                                                         ______________________________________                                        Inhibition of macromolecule synthesis in B16 melanoma cells                             IC.sub.50 (mc/ml)                                                   Compound    DNA         RNA     Protein                                       ______________________________________                                        BU-3862T    0.10        11      0.060                                         Mitomycin C 1.6         11      60                                            ______________________________________                                    

                  TABLE 6                                                         ______________________________________                                        Antitumor activity of BU-3862T against B16 melanoma (ip)                                          Treat-              Body wt.                                                  ment                change                                         Dose       schedule MST*.sup.1                                                                          T/C  on                                    Compound (mg/kg/day)                                                                              (ip)     (day) (%)  day 5 (g)                             ______________________________________                                        BU-3862T 8.0        Q4D × 3                                                                          20.0  167*.sup.2                                                                         -2.3                                           4.0        "        19.0  158*.sup.2                                                                         -1.3                                           2.0        "        18.0  150*.sup.2                                                                         -0.5                                           1.0        "        17.0  142*.sup.2                                                                         +0.3                                           0.5        "        15.5  129*.sup.2                                                                         +0 5                                           0.25       "        14.0  117  +0.5                                  Mitomycin C                                                                            2.0        Q4D × 3                                                                          29.0  242*.sup.2                                                                         +0.3                                           1.0        "        18.5  154*.sup.2                                                                         +0.5                                           0.5        "        15.0  125*.sup.2                                                                         +0.5                                           0.25       "        13.0  108  +0.3                                  Vehicle  --         Q4D × 3                                                                          12.0  --   +1.1                                  BU-3862T 2.0         QD × 9                                                                          Tox.  Tox. --                                             1.0        "        14.5   97  -3.3                                           0.5        "        20.0  133*.sup.2                                                                         -2.3                                           0.25       "        20.0  133*.sup.2                                                                         +0.3                                           0.13       "        18.5  123  +0.8                                           0.63       "        18.0  113  +1.3                                  Vehicle  --          QD × 9                                                                          15.0  --   +1.3                                  ______________________________________                                         *.sup.1 Median survival time                                                  *.sup.2 Significant antitumor effect (T/C ≧ 125%)                 

                  TABLE 7                                                         ______________________________________                                        Antitumor activity of diacetyl- and dihydro- BU-3862T                         against B16 melanoma (ip)                                                                         Treat-              Body wt.                                                  ment                change                                         Dose       schedule MST*.sup.1                                                                          T/C  on                                    Compound (mg/kg/day)                                                                              (ip)     (day) (%)  day 5 (g)                             ______________________________________                                        Diacetyl-                                                                              8.0        Q4D × 3                                                                          24.0  141*.sup.2                                                                         -2.0                                  BU-3862T                                                                               4.0        "        23.0  135*.sup.2                                                                         +0.5                                           2.0        "        20.5  121  +1.0                                           1.0        "        20.0  118  +0.3                                           0.5        "        19.0  112  +1.3                                  Dihydro- 8.0        Q4D × 3                                                                          22.5  132*.sup.2                                                                         -1.0                                  BU-3862T                                                                               4.0        "        20.0  118  -0.5                                           2.0        "        21.0  124  +0.5                                           1.0        "        19.5  115  +0.5                                           0.5        "        19.0  112  +0.5                                  Mitomycin C                                                                            2.0        Q4D × 3                                                                          33.0  194*.sup.2                                                                          0.0                                           1.0        "        23.0  135*.sup.2                                                                         +1.0                                           0.5        "        22.0  129*.sup.2                                                                         +0.3                                            0.25      "        20.0  118  +0.3                                  Vehicle  --         Q4D × 3                                                                          17.0  --   +0.8                                  ______________________________________                                         *.sup.1 Median survival time                                                  *.sup.2 Significant antitumor effect (T/C ≧ 125%)                 

                  TABLE 8                                                         ______________________________________                                        Antitumor activity of BU-3862T against L1210 leukemia (ip)                                                         Body wt.                                           Dose*.sup.1                                                                              MST*.sup.2                                                                             T/C    change on                                Compound  (mg/kg/day)                                                                              (day)    (%)    day 5 (g)                                ______________________________________                                        BU-3862T  4.0        Tox.     Tox.   --                                                 2.0        11.5     144*.sup.3                                                                           -3.5                                               1.0        10.0     125*.sup.3                                                                           -2.8                                               0.5         9.5     119    -0.8                                                0.25       9.5     119     0.0                                     Mitomycin C                                                                             2.0        13.0     163*.sup.3                                                                           -0.5                                               1.0        11.0     138*.sup.3                                                                           -0.3                                               0.5        10.5     131*.sup.3                                                                            0.0                                                0.25      10.0     125*.sup.3                                                                           +0.8                                     Vehicle   --          8.0     --     +1.1                                     ______________________________________                                         *.sup.1 Q3D × 3, ip for BU3862T and QD × 3, ip for mitomycin      *.sup.2 Median survival time                                                  *.sup.3 Significant antitumor effect (T/C ≧ 125%)                 

                  TABLE 9                                                         ______________________________________                                        Antitumor activity of BU-386ZT against P388 leukemia (ip)                                                          Body wt.                                           Dose*.sup.1                                                                              MST*.sup.2                                                                             T/C    change on                                Compound  (mg/kg/day)                                                                              (day)    (%)    day 5 (g)                                ______________________________________                                        BU-3862T  4.0        11.0     105    -2.3                                               2.0        11.0     105    -2.8                                               1.0        11.5     110    -0.8                                               0.5        13.0     124    -0.3                                                0.25      12.5     119    +0.8                                     Mitomycin C                                                                             2.0        17.0     162*.sup.3                                                                           +1.5                                               1.0        15.0     143*.sup.3                                                                           +1.8                                               0.5        13.0     124    +1.8                                                0.25      13.0     124    +2.3                                     Vehicle   --         10.5     --     +2.4                                     ______________________________________                                         *.sup.1 Q4D × 3, ip                                                     *.sup.2 Median survival time                                                  *.sup.3 Significant antitumor effect (T/C ≧ 125%)                 

As indicated by the data shown above, BU-3862T and its dihydro anddiacetyl derivatives are useful as antitumor agents for inhibition ofmammalian malignant tumors such as B16 melanoma.

The invention includes within its scope pharmaceutical compositionscontaining an effective tumor-inhibiting amount of BU-3862T,dihydro-BU-3862T or diacetyl-BU-3862T in combination with an inertpharmaceutically acceptable carrier or diluent. Such compositions mayalso contain other active antitumor agents and may be made up in anypharmaceutical form appropriate for the desired route of administration.Examples of such compositions include solid compositions for oraladministration such as tablets, capsules, pills, powders and granules,liquid compositions for oral administration such as solutions,suspensions, syrups or elixers and preparations for parenteraladministration such as sterile solutions, suspensions or emulsions. Theymay also be manufactured in the form of sterile solid compositions whichcan be dissolved in sterile water, physiological saline or othersuitable sterile injectable medium immediately before use.

For use as an antitumor agent, optimal dosages and regimens of BU-3862Tor its dihydro or diacetyl derivative for a given mammalian host can bereadily ascertained by those skilled in the art. It will, of course, beappreciated that the actual dose of compound used will vary according tothe particular composition formulated, the mode of application and theparticular situs, host and disease being treated. Many factors thatmodify the action of the drug will be taken into account including age,weight, sex, diet, time of administration, route of administration, rateof excretion, condition of the patient, drug combinations, reactionsensitivities and severity of the disease.

The following examples are provided for illustrative purposes only andare not intended to limit the scope of the invention.

EXAMPLE 1 Fermentation of BU-3862T

A well grown agar slant of Streptomyces hygroscopicus, Strain No.P247-71, was used to inoculate a vegetative medium consisting of 3%soybean meal (Nikko Seiyu), 0.5% Pharmamedia (Traders, U.S.A.), 3%glucose, 0.1% yeast extract (Oriental) and 0.3% CaCO₃, the pH beingadjusted to 7.0 before sterilization. The vegetative medium wasincubated at 28° C. for 4 days on a rotary shaker (200 rpm) and 5 ml ofthe growth was transferred into a 500-ml Erlenmeyer flask containing 100ml of the fermentation medium having the same composition as thevegetative medium. The fermentation was carried out at 28° C. for 4 to 5days with shaking on the rotary shaker.

The antitumor antibiotic production in the fermentation broth wasdetermined by in vitro cytotoxic activity against B16 melanoma cells.The fermentation was also carried out in a tank fermentor. A 2-literportion of the vegetative culture by the flask fermentation wastransferred into a 200-liter tank fermentor containing 120 liters of thefermentation medium. Fermentation was run at 28° C. with agitation at250 rpm and aeration rate of 120 liters per minute. The antitumorantibiotic level reached a maximum of 50 μg/ml after about 90 hoursfermentation.

EXAMPLE 2 Isolation and Purification of BU-3862T

The fermentation broth (23 L, pH 7.4) obtained by the general procedureof Example 1 was separated into the mycelial cake and the supernate byuse of a Sharpless-type centrifuge (Kokusan No. 4A). The mycelial cakewas extracted with methanol (6 L). After removal of the insolubles byfiltration, the methanolic extract was concentrated in vacuo to anaqueous solution. This aqueous solution and the supernate of thefermentation broth were combined and extracted with ethyl acetate (20L). The extract was evaporated to dryness in vacuo yielding 21.1 g ofcrude antibiotic complex. This crude solid was applied on a column ofsilica gel (φ4.0×75 cm) which had been pre-washed with methylenechloride, and was developed by a methylene chloride-methanol mixturewith stepwise increase of methanol concentration (2-10% v/v). The eluantwas monitored by cytotoxicity against B16 melanoma and color reactionwith iodine on a TLC plate. The first iodine positive fractions elutedwith 2% methanol were collected and further purified by Sephadex LH-20chromatography. The purified component was identified as9-methylstreptimidone¹) on the basis of its spectral data. The secondiodine positive fractions eluted with 5% methanol were collected andevaporated in vacuo to afford semi-pure solid of BU-3862T. This wasfurther chromatographed on silica gel using ethyl acetate-methanolmixture. Elution with the mixture of 50:1 v/v ratio gave activefractions which showed strong cytotoxicity against B16 melanoma. Afterconcentration in vacuo, the residue was further purified by SephadexLH-20 chromatography with methanol elution to afford a homogeneous solidof BU-3862T (341 mg).

EXAMPLE 3 Preparation of Diacetyl-BU-3862T

BU-3862T (10 mg) was stirred with acetic anhydride (0.1 ml) and drypyridine (0.5 ml) for 18 hours at room temperature. The reaction mixturewas diluted with ethyl acetate (10 ml), and the solution was washed withdilute HCl (10 ml) and then water (10 ml). The organic solution wasdried over Na₂ SO₄ and evaporated in vacuo to give oily diacetylBU-3862T (13 mg). Physico-chemical properties are listed in Tables 9 and10 below.

                                      TABLE 9                                     __________________________________________________________________________    Physico-chemical Properties of BU-3862T Derivatives                                      Diacetyl-BU-3862T                                                                        Dihydro-BU-3862T                                                                           Tetrahydro-BU-3862T                        __________________________________________________________________________    Nature     Colorless sticky solid                                                                   Colorless sticky solid                                                                     Colorless sticky solid                     SIMS m/z   483 (M + H)+                                                                             401 (M + H)+ 403 (M + H)+                               EIMS m/z   423 (M--COOCH.sub.3)+                                                                    370 (M + H--CH.sub.2 OH)+                                                                  372 (M + H--CH.sub.2 OH)+                             430        299          299                                                   256        214          214                                                   127        127          127                                        IR νKBr cm.sup.-1                                                                     3300       3300         3300                                       max        3070       3070         3070                                                  2950       2950         2950                                                  1750       1720         1710                                                  1650       1640         1640                                                  1550       1530         1530                                                  1240       1050         1050                                                  1040                                                               TLC, SiO.sub.2  Rf                                                                       0.78       0.37         0.21                                       CH.sub.2 Cl.sub.2 --MeOH (9:1)                                                __________________________________________________________________________

                  TABLE 10                                                        ______________________________________                                        'H-NMR Data of Bu-3862T and Its Derivatives (400 MHz in                       CDCl.sub.3)                                                                    ##STR8##                                                                                       Diacetyl- Dihydro-                                                                              Tetrahydro-                               Position No.                                                                          BU-3862T  BU-3862T  BU-3862T                                                                              BU-3862T                                  ______________________________________                                         1      3.75 (d)  4.01 (d)  3.73 (d)                                                                              3.74 (dd).sup.a)                                  4.21 (d)  4.87 (d)  4.21 (d)                                                                              3.90 (m)                                   2        --        --        --    3.15 (m)                                   4      4.61 (ddd)                                                                              4.61 (ddd)                                                                              4.51 (m)                                                                              4.58 (m).sup.b)                            5      7.03 (d)  6.53 (d)  7.10 (d)                                                                              7.31 (d)                                   7      4.48 (ddd)                                                                              4.70 (ddd)                                                                              4.49 (ddd)                                                                            4.54 (m).sup.b)                            8      6.48 (d)  6.22 (d)  6.51 (d)                                                                              6.52 (d)                                  10      2.21 (t)  2.21 (t)  2.22 (t)                                                                              2.24 (t)                                  11      1.60 (m)  1.59 (m)  1.60 (m)                                                                              1.61 (m)                                  12      1.28 (m)  1.28 (m)  1.28 (m)                                                                              1.29 (m)                                  13      1.16 (m)  1.18 (m)  1.16 (m)                                                                              1.19 (m)                                  14      1.52 (m)  1.52 (m)  1.52 (m)                                                                              1.52 (m)                                  15      0.86 (d)  0.86 (d)  0.85 (d)                                                                              0.86 (d)                                  16      3.12 (d)  3.09 (d)  3.10 (d)                                                                              3.81 (dd).sup.a)                                  3.35 (d)  3.38 (d)  3.31 (d)                                                                              3.90 (m)                                  17      2.08 (dd) 2.07 (dd)                                                           2.59 (dd) 2.60 (dd) 1.28 (m)                                                                              1.29 (m)                                  18        --        --      1.66 (m)                                                                              1.70 (m)                                  19      1.75 (s)  1.76 (s)  0.94 (d).sup.c)                                                                       0.96 (d).sup.d)                           20      4.79 (brs)                                                                              4.80 (brs)                                                                              0.95 (d).sup.c)                                                                       0.97 (d).sup.d)                                   4.83 (brs)                                                                              4.88 (brs)                                                  21      3.58 (dd) 4.18 (dd) 3.58 (dd)                                                                             3.59 (dd)                                         4.02 (dd) 4.37 (dd) 4.03 (dd)                                                                             4.06 (dd)                                 22      0.86 (d)  0.86 (d)  0.85 (d)                                                                              0.86 (d)                                  OCOCH.sub.3       2.05, 2.09                                                  ______________________________________                                         .sup.a), b), c) and .sup.d) indicate pairs of assignments that may be         interchanged                                                             

EXAMPLE 4 Preparation of Dihydro- and Tetrahydro-BU-3862T

Bu-3862T (30 mg) dissolved in methanol (10 ml) was hydrogenated underatmospheric pressure in the presence of 20% Pd/C (15 mg) for 20 hours.The reaction mixture was filtered and the filtrate was evaporated underreduced pressure to afford a mixture of two hydrogenation products (27mg). They were separated by preparative TLC (SiO₂, CH₂ Cl₂ --MeOH═9:1v/v) and purified by Sephadex LH-20 chromatography to obtain dihydro(13.4 mg) and tetrahydro BU-3862T (4.7 mg). Physico-chemical propertiesare shown in Tables 9 and 10 above.

We claim:
 1. A process for producing BU-3862T of the formula ##STR9##which comprises cultivating Streptomyces hygroscopicus ATCC 53709 in anaqueous nutrient medium containing assimilable sources of carbon andnitrogen under submerged aerobic conditions until a recoverable amountof BU-3862T is produced by said organism in said culture medium and thenrecovering said BU-3862T from the culture medium.